Diagnostic preparation and process for the detection of acetylmethylcarbinol



Sept. 12, 1967 G. 1.. EVANS ETAL 3,341,427 DIAGNOSTIC PREPARATION AND PROCESS FOR THE DETECTION OF ACETYLMETHYLCARBINOL Filed Jan. 22, 1965 BARRIER MIGRATION ZONE REAGENT ZONE (SOLUTION "A") BARRIER MEDIUM ZONE (SOLUTION "B) is INVENTOR GEORGE L. EVANS CHARLES I. HELLER BENJAMIN S. SCHWARTZ ATTORNEY United States Patent 3,341,427 DIAGNOSTIC PREPARATION AND PROCESS FOR DETECTION OF ACETYLMETHYLCARBI- George L. Evans, Hopatcong, and Charles I. Heller and Benjamin S. Schwartz, Livingston, N.J., assignors to Warner-Lambert Pharmaceutical Company, Morris Plains, N.J., a corporation of Delaware Filed Jan. 22, 1965, Ser. No. 427,272 4 Claims. (Cl. 195-1035) This invention relates to a novel composition for the a rapid detection of acetylmethylcarbinol (AMC) produced by microorganisms and relates more particularly to a composition for the rapid differentiation between AMC producing Enterobacteriaceae and non-AMC producing Enterobacteriaceae.

The production and the subsequent detection of acetylmethylcarbinol (AMC) from glycolysis products by certain organisms of the Enterobacteriaceae forms the basis of the Voges-Proskauer or V-P test. This test is a wellknown laboratory diagnostic method for the difi'erentiation and identification of the various organisms which belong to this group. For example, two organisms of this coliform group, i.e., Aerobacter and Klebsiella, produce acetylmethylcarbinol and on this basis may be distinguished from Escherichia coli and E. fretmdii, which do not. However, to carry out this classical test or its more modern adaptations requires the use of incubation periods of a duration too long for rapid identification of the organisms.

Thus, one of the preferred methods for detecting acetylrnethylcarbinol is the Barritt modification, (J Path. 42:441-454, 1936). In this method the unknown organism isolated in pure culture from a specimen such as stool, blood or urine is grown in a -glucose-peptone-phosphate buffer broth for 48 hours. To 1 ml. of the medium is then added 0.6 ml. 5% l-naphthol in ethanol and 0.2 ml. 40% potassium hydroxide. The development of a pink to red color in 30 minutes to 4 hours indicates the presence of acetylmethylcarbinol (AMC).

Another modification is the Benjaminson method (M. A., dc Guzman, B. C. and A. J. Wei], 1. Bact. 87, 234 235, 1964). In this test, isolates grown on triple sugar iron (TSI) agar slants are suspended in 0.5 ml. of 0.5% aqueous creatine solution and then l-naphthol and 40% KOH are added. A pink to red color which develops in 5 minutes is positive for AMC.

It is obvious that there are many disadvantages to these methods. In the Barritt modification, time of incubation is dependent on the volume of reagents and is critical. In addition, pure cultures must be grown in specialized media following primary isolation of the culture. In the method of Benjaminson et al., organisms can be taken directly from a TSI agar slant only if acid has been produced on the slant. The use of organisms from a TSI slant is considered advantageous since this media is usually incorporated into the scheme for separation of the Enterobacteriaceae. However, if acid is not produced .on the slant the testing of these non-lactose fermentors becomes impractical. Hence, the test is limited in practice to Aerobacter-Klebsiella group with the exclusion of other AMC producers.

In all bacterial infections the rapid and accurate identification of the infectious agent is of paramount importance so that proper therapy can be promptly initiated. The above-described tests are obviously impractical when rapid diagnosis is essential. In addition, the necessity for the preparation of the various specific reagents required for the test further hampers the ability to achieve rapid identification.

Accordingly, a'primary object of this invention is to provide a simple and stable composition for the rapid detection of AMC produced by micro-organisms.

A further object of this invention is to provide a simple and rapid process for separating AMC producing organisms from non-AMC producing organisms.

Another object of this invention is to provide a unitary composition containing all the reagents except alkali hydroxide for the V-P test.

Yet another object of this invention is to provide a unitary composition suitable for detecting AMC and having enhanced stability.

Other objects and advantages will become apparent from the following detailed description.

We have now found that the aforementioned objects are fulfilled by providing a strip of a bibulous material which is impregnated over at least a portion of its area with a reagent system comprising l-arginine and anaphthol and over another portion is impregnated with a nutrient medium system comprising nutrients such as brain-heart infusion broth, trypticase and d-glucose and which is adapted to support the growth of microorganisms and to enhance the production of AMC by the growing microorganisms. The concentration of brain-heart infusion and trypticase in the nutrient medium is not critical at one end with a nutrient medium and this area is indicated as being the medium zone in the drawing. The nutrient medium is applied as an aqueous solution. After the solvent has evaporated the hydrophobic material is applied adjacent to this zone and the area carrying this material is further identified in the drawing as the barrier zone. Hydrophobic barrier materials such as lacquer or a chrome complex of the formula:

-.two ingredients. should be applied in the relative proportions of about 10 parts by weight a-naphthol to about 1 part by weight of l-arginine. A further barrier zone may be applied above the reagent zone, as shown in the drawing, in order to prevent excessive migration of the reagent and diffusion of color formed in positive test.

Suitable bibulous materials which can be employed as carriers are those materials which by means of capillary action are able to draw a liquid upward and materials 3 such as filter paper, felt, porous ceramic strips, woven or matted glass fiber and the like are suitable.

The bibulous material is normally cut into narrow strips to facilitate packaging in small bottles or other containers from which they may be dispensed as needed.

In use, the end of the bibulous material impregnated with the culture medium is allowed to come in contact with a suspension of a pure culture of unknown bacteria contained in a test tube and, while in this position in the tube, both the tube and strip are incubated together at 37 C. for 4 to 5 hours. The suspension of bacteria need not be grown in a special media or incubated for long duration for this test. The strip is then removed and a few drops of 40% alkali hydroxide are added to the tube. The reagent zone of the bibulous material is then lowered into the culture and allowed to come into contact with the alkali treated suspension.

An alternative method for developing the color comprises introducing a few drops of the alkali hydroxide solution into the test tube after incubation without wetting the reagent zone of the test strip. The entire system is then gently mixed after which the reagent zone is allowed to come in contact with the suspension. A positive test for the presence of AMC is indicated by a pink to cherry red color which is quite prominent in the migration zone. This cherry red color appears in about 5 to 30 minutes.

The advantages and convenience of the above-described invention are quite outstanding in view of the fact that the test for AMC can be carried out under conditions employing a very much shorter incubation time than pjreviously considered necessary. In addition, this test has also been found to be more sensitive for detecting AMC than conventional techniques.

The following examples are included in order further to illustrate the invention.

EXAMPLE 1 A. Preparation of reagent system (Solution A) One gram of l-arginine is dissolved in about ml. of water. To this is added with stirring a solution of 10 grams a-naphthol in 30 ml. of 95% ethanol.

B. Preparation of nutrient medium (Solution B) 37 grams of dehydrated brain-heart infusion broth, 10 grams of trypticase, 37.5 grams of d-glucose is dissolved together with the aid of gentle heating until the solution is complete.

C. Application to bibulous material In preparing the test strip the initial step is the application of two barrier zones which act to prevent the migration of aqueous solutions which are subsequently applied. These barrier zones are applied to a suitable filter paper such as Eaton-Dykman #55053 in the positions indicated in the accompanying drawing by employing a solution of a lacquer which produces a water-impervious film-forming coat. After allowing the lacquer solution to dry, Solution A and Solution B prepared as described above are then applied to the paper also in the positions indicated in the diagram. The zones thus formed are then allowed to dry.

EXAMPLE 2 Use of the reagent strips 1 to 2 loopfuls of an unknown bacterium which was previously grown on agar slant is suspended in 0.4 ml. of isotonic saline. A test strip prepared according to Example 1 is placed in the tube with the bottom or medium zone immersed in the suspension of the organism. The test strip and the organism suspension are incubated at 37 C. for about 4 to 5 hours. After the incubation period, the paper strip is removed and 2 drops of 40% by weight aqueous potassium hydroxide solution are added to the test tube. The potassium hydroxide treated bacterial suspension is then brought into contact with the reagent zone of the test strip. Alternatively, the paper may be allowed to remain in the test tube after incubation and when the several drops of 40% potasium hydroxide are added to the tube care is taken to avoid wetting the reagent zone. The tube is gently agitated and the reagent zone is then allowed to come in contact with the suspension. A positive test for the presence of acetylmethylcarbinol is indicated by the formation of a pink to red color.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of our invention.

Having described our invention, what we desire to secure by Letters Patent is:

1. A diagnostic preparation for the rapid and positive detection of acetylmethylcarbinol which comprises a strip of a bibulous material impregnaated with a medium zone comprising brain-heart infusion, trypticase and glucose; a reagent zone comprising l-arginine and a-naphthol and a hydrophobic barrier zone separating said medium Zone and reagent zone.

2. A diagnostic preparation for the rapid and positive detection of acetylmethylcarbinol which comprises a strip of a bibulous material impregnated with a medium zone comprising brain-heat infusion, tryticase and glucose; a reagent zone comprising l-arginine and a-naphthol; a first hydrophobic barrier zone separating said medium zone and reagent zone and a second hydrophobic barrier Zone at a suitable distance above the reagent zone.

3. A diagnostic preparation for the rapid and positive detection of acetylmethylcarbinol which comprises a strip of a bibulous material impregnated with a medium zone comprising about 5 parts to about 25 parts by weight of brain-heart infusion, 1 part to 10 parts by weight of trypticase and about 25 parts by weight of glucose; a reagent zone in which the reagent comprises about 10 parts by weight of a-naphthol and 1 part by weight of l-arginine; a first hydrophobic barrier zone separating said medium zone and reagent zone and a second barrier zone at an appropriate height above said reagent zone.

4. Process for the detection of acetylmethylcarbinol produced by micro-organisms which comprises allowing the medium zone of a test strip as defined in claim 3 to come in contact with a suspension of an unknown bacteria for about 4 to 5 hours at 37 0., adding an aqueous solution of an alkali metal hydroxide to said bacterial suspension and allowing the alkali hydroxide treated bacterial suspension to come in contact with the reagent zone of said test strip.

References Cited UNITED STATES PATENTS 3,011,874 12/1961 Deutsch 23253 OTHER REFERENCES Difco Manual, Ninth Ed., Difco Laboratories Inc.. Detroit, Mich., 1953, pages 54 and 55.

ALVIN E. TANENHOLTZ, Primary Examiner. 

1. A DIAGNOSTIC PREPARATION FOR THE RAPID AND POSITIVE DETECTION OF ACETYLMETHYLCARBINOL WHICH COMPRISES A STRIP OF BIBULOUS MATERIAL IMPREGNAATED WITH A MEDIUM ZONE COMPRISING BRAIN-HEART INFUSION, TRYPTICASE AND GLUCOSE; A REAGENT ZONE COMPRISING 1-ARGININE AND A-NAPHTHOL AND A HYDROPHOBIC BARRIER ZONE SEPARATING SAID MEDIUM ZONE AND REAGENT ZONE. 